We are broadly interested in post-transcriptional gene regulation and our current focus is on the mRNA 3′ end processing. The 3′ ends of most eukaryotic mRNAs are formed by an endonucleolytic cleavage and the subsequent addition of a string of adenosines. Interestingly, the transcripts of 30-70% of genes in all eukaryotes have alternative 3′ ends that are formed by cleavage/polyadenylation at different sites, a phenomenon called mRNA alternative polyadenylation (APA). APA not only expands the proteomic and functional diversity, but also plays important roles in gene regulation. Deregulation of mRNA 3′ processing and APA have been implicated in a wide spectrum of human diseases. However, it remains poorly understood how poly(A) sites are recognized and how their recognition is regulated. Our goal is to decipher the rules that govern poly(A) site choice, or the “polyadenylation code”, by using a combination of biochemical, genomic, and genetic approaches.
- Characterization of the mRNA 3′ processing machinery.
Previously we have purified the human mRNA 3′ processing complex in its active and intact form (Shi et al., Mol Cell 2009). Surprisingly, this complex consists of more than 80 proteins, including the core 3′ processing factors and many peripheral factors that may couple mRNA 3′ end formation to other cellular processes. Currently we are carrying out proteomic, structural and functional analyses to understand the inner workings of this amazing molecular machine.
Electron microscopy images of purified human mRNA 3′ processing complex (Shi et al., Mol Cell 2009)
- Regulation of mRNA alternative polyadenylation (APA).
Recently we discovered that the core mRNA 3′ processing factor CstF64 is an important global regulator of APA (Yao et al, PNAS 2012). This was accomplished by mapping the CstF64-RNA interactions at the transcriptome level and characterizing global APA changes upon CstF64 depletion. By using similar approaches, we are currently in the process of identifying and characterizing other APA regulators.
- mRNA APA regulation in stem cells and cancer.
We have recently developed a high throughput sequencing-based method called PAS-seq for quantitatively RNA polyadenylation profiling at the transcriptome level. Using this method, we detected extensive and systematic changes in global APA profile during stem cell differentiation to neurons (Shepard et al., RNA 2011). However, it remains unknown whether and how APA regulation may functionally contribute to stem cell differentiation. Similarly, APA regulation has been implicated in cancer development. We are currently investigating the functional significance of APA regulation in these processes.
Our research is funded by: